Magnetic and Photocatalytic Curcumin Bound Carbon Nitride Nanohybrids for Enhanced Glioma Cell Death [component]

A mesoporous magnetic nanohybrid functionalized with 14 wt% carbon nitride (CN) and loaded with curcumin (Cur) has been developed as a combination platform for photodynamic therapy and magnetic hyperthermia. CN-Cur complexes on the nanoparticle surface facilitate fast charge separation of hole-electrons under blue light irradiation and subsequent singlet oxygen generation. Cur release from the nanoparticle was significant only when exposed to both lysosomal pH (pH=5.2) and an alternating
more » ... magnetic field (AMF). The mesoporous magnetic carbon nitride (MMCN) caused a 350% increase in the level of intracellular ROS as compared to the light exposed untreated control group. The nanohybrid was non-hemolytic and found to be biocompatible with HUVEC cells at concentrations up to 360 µg/mL. A similar concentration under AMF exposure caused a localized temperature rise of 4.2 °C and resulted in a 60% reduction in C6 cell viability. The cancer cell death further increased up to 80% under sequential exposure to light and AMF. The combinatorial treatment exerted significant cytoskeletal and nuclear damage in the cancer cells as assessed by confocal microscopy. The nanohybrid also exhibited relaxivity of 88 mM -1 s -1 , imparting significant T2 weighted contrast to the cancer cells. Multifunctional nanoparticles for biomedical applications have gained much attention in recent decades, especially for cancer management. In this context, magnetic nanoparticles (MNPs) have been extensively used due to their optimal growth characteristics, low cost, ease of synthesis, low toxicity and unique magnetic properties [1] [2] [3] [4] [5] . MNPs have been used for various theranostic purposes such as MRI contrast agents, remote activation of cell functions, imaging, hyperthermia, targeted drug delivery, biosensing, etc. MNPs when exposed to an external alternating magnetic field (AMF) generate heat, which can be used for treatment of cancer [6] [7] [8] [9] . Additionally, the mechanical stress induced by the intracellular oscillation of MNPs when subjected to AMF can cause structural damage to the cells [10]. MNPs exhibit peroxidase-like activity in the acidic environment of the tumor niche, converting H2O2 to hydroxyl radicals ( _ OH) and which subsequently impart damage to vital macromolecular entities such as DNA/proteins leading to cell death [11]. It has been reported that after cellular uptake, MNPs of various size i.e. 20-310 nm do not demonstrate noticeable short-or long-term toxicity [12] [13] [14] [15] , and it has been suggested that they slowly disintegrate into iron ions and are translocated by the divalent metal transporter-1(DMT1) across the endo/lysosomal membranes. Internalized iron is transported to mitochondria for the synthesis of haem or iron-sulfur clusters, which are integral parts of several metalloproteins, and excess iron is stored and detoxified in cytosolic ferritin. [16] [17] [18] . Cancer cell damage can be amplified several fold by conferring photo-responsiveness to MNPs to generate an excess of singlet oxygen species by the clinically established procedure of photodynamic therapy (PDT). PDT is a noninvasive and mild medical technique for anti-cancer therapy that utilizes a photosensitizer to yield reactive oxygen species (ROS) upon illumination of light of a specific wavelength. On light excitation, photosensitizers transfer the absorbed photonenergy to surrounding oxygen molecules, generating free radicals (·O2 − ) or singlet oxygen ( 1 O2) leading to cancer cell death and tissue damage [19, 20] . A host of first and second generation organic photosensitizers including BODIPY, pPorphyrins, chlorins, phthalocyanin etc., have been reported, however these compounds are susceptible to photobleaching and tend to aggregate resulting in a short life time thereby decreased singlet oxygen yield [21] . Graphitic carbon nitride (g-C3N4) has been used as a photocatalyst due to its adjustable band gap and band position [22] . g-C3N4 has been used for various biomedical applications due to its tailored size, high aqueous dispersity, and low toxicity [23][24][25][26][27]. The two dimensional nanomaterial has been extensively exploited in applications such as water splitting, contaminant degradation, CO2 reduction, and organic synthesis under visible light. The photocatalytic activity of g-C3N4 is restricted due to its rapid electron-hole recombination and lack of prominent absorption in visible region [28][29][30]. Several surface modification methods such as metal-non-metal doping, formation of heterojunctions with other semiconductors, incorporation with carbonaceous materials, copolymerization etc. have been used to enhance the charge separation efficacy and visible light absorption of g-C3N4. Complexing g-C3N4 with photosensitizers such as metalloporphyrin derivatives can enhance its photocatalytic activity [31] [32] [33] . However, utilizing a non-metallic and naturally derived photoactive compound such as curcumin is warranted for therapeutic applications. A potent anti-cancer drug, curcumin has limited utility due to its low bioavailability and high hydrophobicity [34] . Many reports have confirmed curcumin's ability as photosensitizer for generation of different ROS species, such as singlet oxygen and hydroxyl radicals [35] . It is proposed that curcumin would not only amplify the visible light absorption of g-C3N4 but also would accelerate the separation of electron hole pairs. In this work, we report the synthesis of mesoporous magnetic nanoparticles functionalized with carbon nitride (MMCN) for combination therapy. This porous assembly has a large surface area for curcumin loading and Fe release towards photocatalytic and Fenton-based oxidative stress, respectively. Following PDT, AMF exposure not only triggers intracellular release of curcumin from the MMCN but also causes a localized temperature rise inducing cellular damage followed by cancer cell death. Materials and Methods Materials Used Iron (III) chloride hexahydrate (FeCl3·6H2O), iron (II) chloride tetrahydrate (FeCl2·4H2O) and curcumin were obtained from Sigma Aldrich. Ethylene glycol (EG) was purchased from TCI Chemicals. Ethylene diamine (ED) and melamine were purchased from SDFCL. Double distilled (DD) water was used in all synthetic experiments. All the chemicals were of analytical grade and used as received. Synthesis procedure of MMNP/MMCN The MMNP and MMCN particles were prepared through a modified solvothermal method reported by Mohapatra et al. [36] . Briefly, 1.0812 g of FeCl3·6H2O (4mM) and 0.3976 g of FeCl2·4H2O (2 mM) (2:1 molar weight ratio) were dissolved in 8 mL of ED and 30 mL of EG. The solution was stirred vigorously and heated at 50 °C to obtain a homogenous golden brown solution. For the synthesis of the MMCN sample, 0.3 g of melamine was added at this step to form a clear solution. The mixture was transferred to a Teflon-lined stainless steel autoclave (50mL capacity). The autoclave was heated to 200 °C and for 24 h, and then cooled to room temperature. The magnetic particles were separated from the solvent by using a permanent magnet and rinsed 4-5 times with ethanol to remove unreacted species. The washed product was dried in an oven at 60 °C and stored for further use. Synthesis procedure of CN Melamine was used as the source for the synthesis of carbon nitrides nanoparticles by the thermal polymerization method. Briefly, 0.3 g melamine was ground thoroughly and closely packed in an alumina crucible. The crucible was heated at 200°C for 1 h in an air atmosphere with a heating rate of 5°C/min. The as synthesized g-C3N4sheets were 3-4 times washed with MQ water by centrifugation to remove the unreacted product. The heating of melamine at 200°C for 1 h would lead to incomplete polymerization. A pale off-white colored powder was obtained after drying. Structural Characterization X-ray diffraction (XRD) analysis was carried out on a Bruker D8 Advances X-ray diffractometer with Cu Kα (λ = 1.5406 Ǻ) radiation in the 2θ range of 10 to 80 degrees under an accelerating voltage and applied current of 40 kV and 25 mA, respectively. Structural characterization of the particles was performed using a Transmission Electron Microscope (TEM) (JEOL JEM-2100) microscope at an accelerating voltage of 200 kV. The samples were dispersed in water and drop cast on a 400 mesh copper grid coated with perforated carbon film. Elemental mapping of the sample was obtained on the TEM instrument. The surface charge measurements of MMCN in varying pH were performed with a Zetasizer Nano ZSP (Malvern) instrument. The Brunauer-Emmett-Teller (BET) surface area of the particles was measured on an Autosorb iQ2 instrument, (Quantachrome). Thermogravimetric analyses (TGA) were performed under nitrogen atmosphere using a Perkin Elmer STA 8000. All UV-Visible spectra were recorded at room temperature with a Shimadzu UV-2600 spectrophotometer using a quartz cuvette (1cm path length). Magnetic Characterization The magnetic properties of MMNP and MMCN were measured using a vibrating sample magnetometer (VSM, Model 7410, Lake Shore) at room temperature. The magnetic hyperthermia efficiency of the MMCN was measured by the DM2 applicator DM100 system (nB nanoscale Biomagnetics, Zaragoza, Spain). The particles were dispersed in water and subjected to an alternating current (AC) magnetic field (B) at f = 405 kHz (Field = 168 Oe). Generation of Free Hydroxyl Radicals (•OH)/ Fenton Reaction To study the peroxidase-like behavior of the synthesized MMCN 1mg NPs were dispersed in 1 mL of acidic buffer (pH 4.4) to facilitate the release of ferrous (Fe 2+ ) ions. The MMCN solution was incubated with H2O2 and N, N-diethyl-p-phenylenediamine (DPD, 10 mM) for 60 min. The UV-Vis spectra were recorded at different time intervals [37] . Generation of Superoxide radicals/ Photoreaction/ Singlet Oxygen Detection Cur-MMCN was dispersed in water containing the singlet oxygen sensor 1, 5dihydroxynapthalene (1mM DHN) and mixed thoroughly [38] . The solution was exposed to blue LED light for different time intervals of 0-20 min. UV-vis absorption spectra of the supernatant were recorded on a spectrophotometer. At different time intervals i.e. 0-20 min, the sample was placed under a strong magnet to separate the supernatant. The DHN consumption was monitored by observing the decrease in the absorption at around 300 nm with increased irradiation time. CN and curcumin alone were taken as controls. The same experiment was repeated in the absence of light to monitor the effect of light for the production of superoxide radicals. In vitro drug loading and release behavior studies 10 mg of particles (MMNP, MMCN and CN) were dispersed in 1 ml of double distilled water and sonicated for 10 min. A fixed volume (100 µL) of curcumin (Cur) was added to each suspension of particles from a stock (10mg/mL) in methanol. After overnight shaking at 180 rpm, the drug loaded particles (MMNP and MMCN) were separated magnetically from the free drug molecules. Drug loaded CN particles were separated by centrifugation at 7200 rpm for 10 min. The process was repeated another two times to ensure that weakly adsorbed drugs on the surface were removed. The drug loaded particles were stored at 4 °C until further use. To examine the loading efficiency, a standard curve was plotted with known concentrations of Cur dissolved in buffers at pH 7.4 and pH 5.2. The concentration of drug in the supernatant was measured from the absorbance at 425 nm against a standard curve. The percentage of drug entrapped in the NPs was calculated using the following relation. ( )% = ( − )/ * Where Di=Amount of drug added initially, Ds=Amount of drug in the supernatant. The drug release behavior of the finally prepared Cur loaded MMCN nanoparticles were studied in buffered solution at physiological pH (7.4) and acidic lysosomal pH (5.2). 5 mg of all the drug loaded nanoparticles (MMNP-Cur, MMCN-Cur and CN-Cur) were dispersed in 5 mL of PBS (pH 7.4) and acetate buffer (pH 5.2). Both the buffers were added with 0.5% sodium dodecyl sulfate (SDS) to facilitate solubility of Cur [39]. The samples were distributed equally in 10 parts and kept in a shaker incubator at 37 °C. After specific time intervals (0, 0.5, 1, 3, 6, 12, 24, 36, 48 and 60 h) the samples were separated magnetically and the absorbance of the supernatant was recorded. In order to monitor the magnetically triggered controlled release behavior of Cur-MMCN, an AMF of 405 kHz (field strength of 168 Oe) was applied at different time intervals [40]. Cell culture 3-(4,5-dimethyl-thiazol-2yl)-2,5-diphenyl tetrazolium bromide (MTT), bis benzimide (Hoechst 33342) and phalloidin-tetramethyl rhodamine-B-isothiocyanate (Ph-TRITC) conjugates were purchased from Sigma Aldrich. Dulbecco's modified eagle medium (DMEM), antibiotic and antimycotic solution were obtained from Hi-Media Ltd (Mumbai, India). NIH3T3 (mouse embryonic fibroblast) and C6 (rat origin glioblastoma) cell lines were procured from the National Center of Cell Science (NCCS, Pune, India). The cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics antimycotic solution at 37 °C in a humidified incubator containing 5% CO2. Human umbilical vein endothelial cells (HUVEC) (Cat. No. 2517A) were purchased from Lonza and maintained in endothelial growth medium supplemented with Bullet kit (Lonza Cat. No. 3162). All the cells were maintained by sub culturing twice weekly. In vitro biocompatibility The biocompatibility of MMCN, MMNP and CN was evaluated against NIH3T3 cells using an MTT assay. Briefly, 200 µL of cells were seeded in a 96-well flat culture plate at a density of 2.5x10 4 cells per well and cultured for 24 hrs at 37 °C and 5% CO2. To analyze cytocompatiblity, varying concentrations of MMCN, MMNP and CN were added to the cells, and incubated for 48 hours. The biocompatibility of MMCN was studied in HUVEC cells. For this, HUVEC were seeded at a density of 1.5x10 4 cells in a 96 well plate for 48 hrs at 37 °C and 5% CO2. Next day, the media was replaced with fresh media containing different concentrations of MMCN and incubated for 24 h. The cells were washed with pre-warmed 1X PBS thrice to remove traces of sample. 20 µL MTT solutions (5 mg/mL in PBS) diluted with 180 µL media was added to the wells and incubated for 4 h. After 4 h, the plates were centrifuged at 1500 rpm for 5 min at room of AMF. Arrow and shaded area indicates time point and duration (20 minutes) of AMF exposure. Reactive active species (ROS) generation studies Different studies were carried out to examine the mechanism and source of the ROS generation. Peroxides (O2 2− ), hydroxyl radical (HO•) and singlet oxygen (1O 2 ) cause oxidative damage to the cellular constituents and dysfunctions in cell metabolism. Fenton reaction The porous surface of MMCN under acidic milieu releases Fe 2+ ions, which on reaction with hydrogen peroxide can generate highly reactive hydroxyl and superoxide radicals through the Fenton reaction. Cancer cells produce large amounts of hydrogen peroxide obtained from mitochondria generated superoxide ions, a process that is catalyzed by the over-expressed superoxide dismutase (SOD) [64] . The H2O2 in cancer cells is converted to superoxide radicals and reactive hydroxyl ions catalyzed by Fe 2+ ions in the Fenton reaction. A set of controlled experiments were performed to interrogate SOD-like activity of Cur-MMNP. In the presence of acidic medium the MMCN releases Fe 2+ ions. These Fe 2+ ions further dissociate H2O2 into . OH through Fenton's mechanism. N,N-diethyl-p-phenylenediamine (DPD) is selected as an oxidizable substrate to confirm hydroxyl ion generation. In the presence of H2O2, DPD is oxidised to DPD + , which has a strong absorption at 551 nm as shown in the figure (Fig.S5 ) at 0 minutes. As the incubation time with MMCN increased from 0 to 20 min the intensity of this peak decreased. After 60 min of incubation, the peak has largely disappeared thus inferring complete oxidation of DPD confirming conversion of H2O2 to •OH. In the presence of hydrogen peroxide the MMCN produced •OH. In the control experiments, the colour of the solution turned colourless from pink soon after the addition of H2O2 in the presence of DPD. In the case of MMCN this colour change occured over a period of 60 min. Singlet Oxygen Detection studies The ability of Cur-MMCN to generate singlet oxygen under blue light irradiation was studied by UV-Visible spectroscopy. The consumption of DHN by singlet oxygen species was monitored by a decrease in its absorption at 300 nm. It can be inferred from Fig.S6 that after 20 minutes of light irradiation there is a significant decrease in the DHN absorption peak in Cur-MMCN compared to CN alone, Cur alone and Cur-CN. This warrants the potential of the nanohybrid for photodynamic therapy [38] . Biocompatibility and Hemocompatibility Studies of the biocompatibility of MMNP, MMCN and CN were performed to rule out the effect of nanoparticle toxicity against normal cells [65] . The cell viability assay showed more than 80% of mouse embryonic fibroblast NIH3T3 cells remained viable for the concentration ranges of 0-0.5 mg/mL (Fig.S7 ). The compatibility of MMCN was also examined against Human Umbilical Vein Endothelial cells (HUVEC) for 48 hours showing 75±8.4% viability at 360 µg/mL (Fig.4B ). This was further confirmed by analyzing the nuclear and cytoskeletal integrity via Hoechst and Ph-TRITC staining. As seen in Fig.4A , the bright field image depicts the localization of MMCN in the cytoplasm. Hoechst stained nuclei and Ph-TRITC bound F-actin showed overall intactness to the cell confirming no significant toxicity at 360 µg/mL. As blood is the primary contact for any therapeutic material, its hemocompatibility plays a critical role. MMCN showed less than 0.1 % hemolysis up to the tested concentration of 600 µg/mL similar to the negative control (0.9 % NaCl). This is visibly inferred from the clear supernatant of blood treated with various MMCN concentrations in Fig.4C , as compared to the positive control (1% Triton-X100) which showed significant hemoglobin leakage as evident by the reddish supernatant. The results suggest that MMCN possesses non-hemolytic blood compatibility. Figure-4: Confocal microscopic images of Hoechst (H) and Phalloidin-TRITC (T) stained MMCN treated HUVEC (A), Biocompatibility of different concentrations of MMCN against HUVEC (B) and Percentage hemolysis of MMCN at different concentrations incubated with RBCs (C). In vitro cellular uptake Prussian blue staining was performed to visualize the qualitative uptake of MMCN by C6 cells. C6 cells were incubated for 3 and 6 h with 360 μg/mL of MMCN. The intracellular presence of MMCN can be observed in Fig.5A . The results demonstrate that as the time of incubation is increased, the extent of association of the nanoparticles with the cells increased. The microscopic images confirmed MMCN uptake at the 6 th hour of incubation with no significant effect on cellular morphology [66] . The uptake was also quantified by the ICP-MS analysis. MMCN internalization reflected with presence of 1.65 µg Fe/cell after 6 h of incubation as shown in Fig.5A(iv) , confirming a proportional increase in the amount of Fe with respect to time. Intracellular ROS detection Cells with internalized nanoparticles were exposed to the blue light to demonstrate the potential of the nanohybrid for PDT. To study the changes in intracellular ROS, DCFH-DA dye was used. DCFH-DA is oxidized in the presence of ROS to yield a green fluorescent derivative, DCF. Two different concentrations of Cur (5 and 7.5 µM) loaded particles (CN or MMNP or MMCN) were incubated with C6 cells. After 6 hours of incubation, the cells were exposed to blue light for 20 minutes. The study was also carried out both in the absence of blue light under identical conditions. Compared with the control, light irradiation significantly increased ROS levels in the cells treated with Cur or CN (Fig.5B ). MMNP treated cells showed similar ROS both in presence and absence of light, however Cur-MMCN (7.5µM) showed an increase of 350% of ROS as compared to light exposed control cells, which is significantly higher than ROS present in cells treated with CN, Cur and Cur-CN [67]. Figure-5: Prussian blue staining of MMCN treated C6 cells at 0 (i), 3 (ii) and 6 (ii) hours and their corresponding iron content (iv) as analyzed by ICP-MS (A), Percentage intracellular ROS (B) and percentage cell viability (C) of (a)CN, (b)Cur, (c)MMNP, (d)MMCN, (e)Cur-CN, (f)Cur-MMNP, (g)Cur-MMCN treated C6 cells under dark and light conditions. Two tailed p-value <0.05* and p<0.005**. In vitro photodynamic therapy study The phototoxicity of Curcumin loaded MMCN, MMNP and CN was tested at 1, 2.5, 5 and 7.5 µM equivalent concentration of curcumin on C6 cells in the presence and absence of blue light. Equivalent concentrations of MMCN, MMNP and CN were taken as controls. All the formulations exhibited dose dependent phototoxicity. As seen in Fig.5C , cells treated with Cur-MMCN at 7.5 µM concentration, exhibited viability of ~41%, which is considerably higher than that of cells treated with CN (54.56±2.8%) and . It is also worthwhile to note that 7.5 µM Cur inhibited ~50% cells under light irradiation. This needs to be considered in conjuction with the basal drug release of 10 to 16% from Cur-MMCN (up to ~1.24 µM) while transitioning between extracellular pH into the acidic lysosome at the end of 6 h of treatment. The IC50 value of curcumin alone is reported to be more than 25 µM against C6 glioma cells [67]. According to Dhule et al., the IC50 for DMSO-curcumin and liposomal-curcumin is 22.8 μg/ml (62 µM) and 5.4 μg/ml (14.65 µM), respectively, suggesting that curcumin in the dark requires to be in high concentrations to impart toxicity [68]. MMCN alone could not impart effective phototoxicity on the cancer cells possibly due to the low content of CN i.e., ~14% of NP weight which is 8.3 fold lesser than CN alone used in this study. Only in the presence of Cur did the toxicity of MMCN become prominent, possibly due to enhanced rate of recombination of electron-holes under light irradiation. In addition to the photocatalytic anti-cancer effect of Cur-MMCN, the intrinsic presence of H2O2 in cancer cells might also assist in generation of superoxide and hydroxyl radicals catalyzed by Fe 2+ ions via the Fenton reaction. There was no remarkable decrease in viability of nanoparticle treated C6 cells placed in the dark [69]. Photodynamic therapy and Magnetic hyperthermia Treatment (MHT) MMCN show a temperature rise of ~4.2 °C at 360 µg/mL, which is considerably less than required to heat up the bulk medium to hyperthermia temperatures of 42 o C and above (Fig.S4) . However, Villanueva et al. have shown that AMF induced cellular damage resulted in the death of cancer cells treated with silica-coated manganese oxide perovskites even though the temperature rise of the culture medium was only 0.5 °C. Here, the nuclear morphology was not affected for corresponding controls, however, after 24 h post treatment, morphological alterations was observed in the nucleus causing apoptosis and leading to cell death [70] . In another report, dendritic cells treated with COOH and NH2 functionalized magnetite NPs showed a decrease in viability from 90% to 5% with an overall temperature rise of 2 °C [71] . In concordance with these previous observations, as observed in Fig.6A , Cur-MMCN in combination with blue light could effectively kill ~60 % of cells. However, under identical conditions, an additional insult in the form of AMF (f =405 kHz; Field =168 Oe) for 20 minutes post PDT decreased the cell viability further to ~15% when compared to single treatment groups. The caspase 3/7 apoptosis assay showed that the exposure of either light or AMF caused Cur-MMCN-treated cells to undergo ~47% and ~56% (Fig.6B) apoptosis. However, the highest level of cell death (~70%) was observed in the case of Cur-MMCN treated cells exposed to both light and AMF. These results suggest that the increase in ROS generation during AMF exposure is capable of triggering the apoptotic pathway leading to cancer cell death. The extent of cellular death is directly proportional to the quantitative apoptotic population [72]. Figure-6: (A) Percentage cell viability of untreated and MMCN or Cur-MMCN treated C6 cells exposed to either or both of light and AMF, (B) Percentage of apoptotic cells treated with Cur-MMCN followed by exposure to either or both of light and AMF. Two tailed p-value <0.05*. Confocal microscopic analysis of Cur-MMCN treated cells showed intact structural integrity without exposure to light or AMF, similar to that of the untreated control. The cells exposed with either PDT or AMF in the presence of Cur-MMCN demonstrated significant damage to the cytokeleton as shown in Fig.7 (iii & iv) [73]. Fig.7 (v) substantiates the catastrophic cell death caused by the combination of PDT and AMF. Typical features of apoptosis i.e. cell membrane, cytoskeletal and nuclear damage are clearly visible causing total disintegrity in vital structures followed by cell death. Figure-7: Confocal microscopic images of (i) untreated and Cur-MMCN treated C6 cells followed by exposure to (ii) dark, (iii) blue light, (iv) AMF and (v) blue light + AMF stained with Hoechst (H) and Phalloidin-TRITC (T). MR phantom imaging Fig.8A, shows the MRI phantom of MMCN at different concentration i.e., 0 to 0.12 mM of Fe. The plot of transverse relaxivity (1/T2) versus concentration of iron revealed a R2 relaxivity value of ~73 mM -1 s -1 and ~88 mM -1 s -1 for MMNP and MMCN, respectively. The R2 for MMCN is comparable to a commercial iron oxide based contrast agent (85 mM -1 s -1 ) [74] . Agarose immobilized phantoms of C6 cells treated with MMCN showed superior T2 weighted contrast with increasing nanoparticle concentration (Fig.8C ). Figure-8: (A) Relaxivity plot and (B) T2-weighted MR images corresponding to varying iron concentration of MMNP and MMCN, (C) T2 weighted MR images from C6 cells treated with 0 (i), 1 (ii) and 2.5 (iii) μM curcumin equivalent of Cur-MMCN. CONCLUSION Carbon nitride based nanomaterials have emerged as new class of photocatalysts for photodynamic applications. CN has the capacity to be combined with existing biomaterials such as iron oxide to achieve better therapeutic outcomes. A biocompatible and hemocompatible nanohybrid consisting of carbon nitride (CN) functionalized mesoporous iron oxide nanoparticles loaded with curcumin (Cur) was evaluated as a combination platform for photodynamic therapy and magnetic hyperthermia. Due to the rapid charge separation of hole-electron under light irradiation, Cur-CN combination showed superior generation of intracellular ROS in glioma cells. Apart from imparting significant T2 weighted contrast to glioma cells, a combination of mild magnetic hyperthermia and high oxidative stress induced significant cellular damage leading to apoptosis. The study explored for the first time the combination of CN, curcumin and iron oxide for visible light and AMF driven glioma cell killing. However, due to limited light penetration in biological systems, improvement of the proposed nanohybrid to absorb in the NIR-I &II windows is highly warranted for future studies. Graphical abstract
doi:10.1021/acsbiomaterials.9b01224.s001 fatcat:nkimrlx4hba5fbza2owu6qzemq